Haggerty, Catherine L. Project Abstract Pelvic Inflammatory Disease (PID) is the frequent infection and inflammation of the upper genital tract among young women that often results in infertility, chronic pelvic pain, and recurrent PID. PID is a recognized complication of Chlamydia trachomatis and Neisseria gonorrhoeae infections, yet up to 70% of cases are idiopathic. An association between bacterial vaginosis (BV) and PID is recognized, however few studies have examined the specific vaginal bacteria most linked to PID, particularly focusing on vaginal bacteria that may resist lab cultivation. Cross-sectional evidence implicates Mycoplasma genitalium (MG) and the protozoan Trichomonas vaginalis (TV) as potential causes of PID, but prospective studies are lacking. We propose a prospective cohort study of 1,199 women considered at high risk for sexually transmitted infection who were actively followed three years for PID development. We will develop and apply to samples a panel of quantitative (qPCR) assays for suspect pathogens, including newly identified BV-associated bacteria, MG, and TV based on our pilot studies and broad-range 16S rRNA gene PCR with next generation sequencing and shotgun sequencing in the discovery phase. We will apply the NanoString nCounter inflammation panel to endometrial samples to determine whether particular inflammatory genes are overexpressed in samples from women with PID compared to women without PID in a bacterial species-specific fashion. We will complement our human studies with fallopian tube and endometrial organoid models, allowing us to directly assess how candidate pathogens interact with tissues of the female upper reproductive tract to induce inflammation. Our aims are to: 1) Develop a panel of candidate microbes for the prediction of PID using broad-range 16S rRNA gene PCR with high throughput sequencing (bacteria) and metagenomics shotgun sequencing (all microbes); 2) Determine whether the presence and quantity of vaginal pathogens in our candidate panel are associated with incident PID using sensitive qPCR, and whether the presence of these candidate pathogens in endometrial tissue is associated with an inflammatory gene signature in patients with PID; 3) Develop a model for the prediction of PID; 4) Determine the population attributable fraction (PAF) of PID due to each pathogen in our panel, CT, and NG; and 5) Determine the inflammatory potential of candidate PID pathogens in fallopian tube and endometrial organoid culture models where microbes are inoculated and inflammatory gene expression is assessed. This will be the first prospective study of PID to apply state-of-the-art qPCR assays and vaginal microbiome profiling, creating the potential for pathogen discovery and imparting a greater understanding of PID etiology. Further, we propose the first study to use fallopian tube organoids and NanoString technology to study PID. Testing for non-gonococcal, non-chlamydial pathogens is not routine, and knowledge gained from our study may identify novel diagnostic targets for PID with the potential to optimize future diagnostic, preventive, and treatment approaches.